PCR – Mac’s Bisulfite-Treated DNA

Per Mac’s request, ran a PCR on a set of bisulfite-treated DNA (in her gDNA 2014 box in small -20C):

  • EV2.16 bisulfite
  • EV2.20 bisulfite
  • EV2.22 bisulfite
  • EV2.24 bisulfite
  • EV2.28 bisulfite
  • EV2.29 bisulfite
  • EV2.32 bisulfite
  • EV2.33 bisulfite

DNA needed to be diluted. Diluted according to this sheet provided by Mac:

http://eagle.fish.washington.edu/bivalvia/070914bisulfite.pdf

NOTE: EV2.28 didn’t have sufficient DNA left to prepare the dilution according to Mac’s sheet. Instead, the remaining volume ofEv2.28 bisulfite DNA (0.5uL) was diluted in a total volume of 2.5uL to maintain the same dilution ratio.

Master mix calcs are here: 20140828 – PCR Mac Bisulfite Samples

Primers used were:

CgBS_733_26796Seq (SRID: 1598)
CgBS_733_26796R_5’biotin (SRID: 1596)

Cycling params:

  1. 1. 95C – 10mins
  2. 2. 94C – 30s
  3. 3. 56C – 30s
  4. 4. 72C – 30s
  5. 5. Repeat steps 2 – 5 44 more times
  6. 6. 72C – 10mins

Results:

Ladder used is O’GeneRuler 100bp DNA Ladder (ThermoFisher).

According to Mac, the expected band size is ~300bp. However, all samples are running at ~150bp. Mac is confused and does not know what to do.

*UPDATE 20140902* – Realized I used the wrong forward primer! Will repeat PCR with correct primer. Wonder if Mac did the same thing…

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